The study of a key gene for proliferation, polarization and cell wall integrity and its relationship with Granulin-like family

Author:

Uxue Perez-Cuesta (ES)

Abstract:

Background: The Afu6g07200 gene was detected as overexpressed in the germination process and encodes a hypothetical protein containing a domain that exhibits 95.4% structural similarity to human Granulin A. This growth factor is known to play a role in proliferation and migration of cells, and its presence is believed to be absent in the fungal kingdom. This study aims to elucidate the precise role of the Afu6g07200 gene in the biology of A. fumigatus and investigate its potential function as a growth factor.

Methods: Using the A. fumigatus ΔakuBku80 (Wt) strain as genetic background, a deletion (Δgrn), its complemented, GFP-tagged strains, and a strain with the human Granulin A sequence were generated using a CRISPR-Cas9 technology. The strains were characterized phenotypically using GMM agar plates supplemented with different stressor compounds as well as antifungal drugs. High resolution and fluorescence microscopy were used for detailed analysis. Transcriptomic analysis was performed using RT-qPCR and virulence was assayed in neutropenic mice model.

Results: The loss of function mutant strain (Δgrn) showed less proliferation, swollen areas along the hyphae and tips, cell vacuolization, and more hyphal death than the Wt. In addition, gene deletion defects included an imbalance between mitotic rate, septation, and branching. Septation in the Δgrn strain started earlier than the Wt, with some septa very close even without nuclei between them, and its hyphae showed more branching. The polarization of the Δgrn strain was affected, showing a zigzag phenotype and microtubule-related genes associated with this phenotype were downregulated in this strain. Furthermore, the cell wall of the deletion strain showed poor organization, including a thinner outer layer and wall-to-membrane spaces. We also confirmed a decrease in the chitin content in the Δgrn strain. These changes may be related to the increased sensitivity to calcofluor white and congo red. Moreover, gen deletion also lead to a decrease in caspofungin resistance. Cell wall affectation also included a down expression of the cell wall integrity pathway genes in the Δgrn strain. Fluorescence microscopy observations suggested that this protein localizes to the plasma membrane of the hyphae within the septa and is capable of being secreted to the media. The localization of the protein depended on the maturity of the cell and the growth conditions. Regarding the virulence, differences between the Wt and the loss of function strain were not detected. Finally it is important to highlight that the replacement of the fungal gene with the human granulin A fully phenocopied the Wt phenotype suggesting that this protein could belongs to granulin-like family.

Conclusions: This study is the first to show that a fungal gene could be integrated to granulin-like family due to its similarities in 3D structure and functions. This granulin is involved in hyphal polarization and proliferation, cell cycle, septation, colony morphogenesis, and cell wall construction of the fungus. Moreover, brings to light the need to study the A. fumigatus hypothetical proteins to discover new genes/proteins important for this pathogenic mold, that could be promising therapeutic targets.

Abstract Number: 53

Conference Year: 2024

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